NEW SYNDROME

Recessive Developmental Delay, Small Stature,Microcephaly and Brain Calcifications With Locus onChromosome 2Anna Rajab,1* Kimberly A. Aldinger,2,3 Hisham Ali El-Shirbini,4 William B. Dobyns,3

and M. Elizabeth Ross5**1Genetic Unit, DGHA, Ministry of Health, Muscat, Sultanate of Oman2Committee on Neurobiology, University of Chicago, Chicago, Illinois3Department of Human Genetics, University of Chicago, Chicago, Illinois4Pediatric department, Ibri Regional Hospital, Sultanate of Oman5Laboratory of Neurogenetics and Development, Weill Medical College of Cornell University, New York, New York

Received 9 May 2008; Accepted 15 October 2008

Two interrelated Omani families are described with eight

children manifesting a genetic disorder with widespread brain

calcifications. Brain imaging showed extensive scattered cal-

cifications of basal ganglia and cortex, suggesting possible

Aicardi–Goutieres syndrome (AGS) or Coats’ Plus syndrome.

However, the clinical features in the present families diverge

substantially from these two conditions. Growth delay, mild

developmental delay, and poor school performance were present

in all affected individuals, but progressive deterioration of

neurological function was not apparent, nor were there signifi-

cant cortical whitematter disease or retinopathy. Genome-wide

linkage and fine-mapping analyses of the extended family mem-

bers and affected individuals indicate a genetic locus for this

disorder on Chromosome 2 with a LOD score of 6.17. The

Chromosome 2 locus is novel and the clinical presentation

displays features distinguishing the condition from either

Coats’ or AGS, making this a new variant or possibly a new

disorder of inherited brain calcification. � 2009 Wiley-Liss, Inc.

Key words: brain calcifications; hydrocephalus; autosomal re-

cessive inheritance; microcephaly; developmental delay; Aicar-

di–Goutieres syndrome; coats’ plus syndrome

INTRODUCTION

More than 50 acquired and familial conditions that involve bilateral

calcium accumulation in the brain parenchyma, sometimes re-

ferred to as brain calcinosis, have been reported [Baba et al., 2005].

In children, intracerebral calcifications are most often acquired in

association with intrauterine infections, intrauterine or early post-

natal hemorrhage, or neoplasia. Some features of early onset

cerebral calcifications may be shared between inherited and ac-

quired conditions, but the appearance of multiple affected indi-

viduals in a single family assures an underlying genetic cause.

Many hereditary forms of brain calcinosis involve the basal

ganglia, but only rarely do they involve both deep nuclear structures

and cortical grey matter. Several of them have overlapping pheno-

types, and probably represent examples of the same entity. Several

of the familial forms of brain calcifications may be recognized by

their associated endocrinopathies, which are characterized by

abnormalities of calcium, phosphorous, and parathyroid hormone

(PTH). These include hypoparathyroidism [Ahn et al., 1986] and

pseudohypoparathyroidism (Albright hereditary osteodystrophy)

[Patten et al., 1990; Juppner et al., 1998]. Other metabolic disorders

associated with brain calcification include carbonic anhydrase II

deficiency (CAII) [Sly et al., 1985], Krabbe disease [Farley et al.,

1992], dihydropteridine reductase deficiency [Woody et al., 1989],

Grant sponsor: NIH; Grant number: RO1 NS058721; Grant number: P01

NS048120.

*Correspondence to:

Dr. Anna Rajab, Consultant Clinical Geneticist, P.O. Box 880, Muscat 113,

Sultanate of Oman. E-mail: drarajab@omantel.net.com

**Correspondence to:

Dr. M. Elizabeth Ross, Weill Cornell Medical College, New York, NY 10065.

E-mail: mer2005@med.cornell.edu

Published online 20 January 2009 in Wiley InterScience

(www.interscience.wiley.com)

DOI 10.1002/ajmg.a.32630

How to Cite this Article:Rajab A, Aldinger KA, El-Shirbini HA,

Dobyns WB, Ross ME. 2009. Recessive

developmental delay, small stature,

microcephaly and brain calcifications with

locus on chromosome 2.

Am J Med Genet Part A 149A:129–137.

� 2009 Wiley-Liss, Inc. 129

and mitochondriopathies including MELAS and MERRF [Hirano

and Pavlakis, 1994].

Another group of brain calcinoses is associated with immune

system disorders and systemic involvement. Examples include

systemic lupus erythematosis (SLE) [Baba et al., 2005], IgG

(lambda-M) proteinemia, and autoimmune polyglandular syn-

drome (AIRE) [Baumert et al., 1993]. A significant member of this

class is Aicardi–Goutieres syndrome (AGS), a genetically hetero-

geneous autosomal recessive encephalopathy associated with CSF

lymphocytosis and elevated alpha-interferon [Aicardi and Gou-

tieres, 1984; Lebon et al., 1988; Tolmie et al., 1995].

A similar genetic syndrome distinguished by retinopathy and

extensive cerebral calcification is cerebroretinal microangiopathy

with calcifications and cysts (CRMCC) or ‘‘Coats’ plus’’ syndrome

[Tolmie et al., 1988; Crow et al., 2004; Briggs et al., 2008]. Other rare

causes of intracerebral calcification have been reported such as the

pseudo-TORCH syndrome, a very rare autosomal recessive disor-

der that resembles intrauterine infections, consisting of severe

postnatal microcephaly, seizures, and profound mental retarda-

tion, fronto-parietal polymicrogyria, and a distinct pattern of

frontal predominant, band-like intracranial calcifications [Burn

et al., 1986; Reardon et al., 1994; Briggs et al., in press]. Brain

calcinosis has also been reported with prominent extrapyramidal

movement disorders, as in autosomal dominant dystonia or idio-

pathic basal ganglia calcification (Fahr Disease) [Geschwind et al.,

1999]. In still other brain calcification syndromes, bone dysplasia

may be prominent as in Raine syndrome or polycystic lipomem-

branous osteodysplasia [Kan and Kozlowski, 1992; Al Mane et al.,

1996].

Here we describe a novel autosomal recessive syndrome con-

sisting of early developmental delay, small stature, poor school

performance, and extensive brain calcifications in eight individuals

from two consanguineous and interrelated sibships, whose clinical

features differed from described brain calcinoses. Linkage analysis

mapped the causative locus to 2q36.2, indicating a new gene

associated with intracerebral calcification.

PATIENTS AND METHODS

Clinical StudiesTwo Omani families from the same tribal unit were studied. The

two couples and eight affected children were examined by A.R. and

M.E.R. and the brain imaging results were reviewed independently

by M.E.R. and W.B.D. Plain skull films were obtained on all affected

children and calcifications were apparent. Population based growth

curves have not been established for Oman. Experience indicates

that head circumferences are equivalent to those published in the

UK and US, but height and weight curves of Omani children

between the ages of 2 and 20 years are shifted approximately

�1.5 SD, so that the 50th centile for Oman corresponds to

�1.5 SD on growth curves in the West. Therefore, percentiles here

were based on the growth curves published by the Center for Disease

Control (CDC) and adjustments for height and weight were also

estimated for the Omani population. Brain computerized tomog-

raphy (CT) was performed and reviewed on all affected individuals

and parents and magnetic resonance imaging (MRI) was obtained

on the eldest daughter in family 1 at age 18. Medical records were

reviewed by A.R. and skeletal surveys were reviewed by M.E.R. with

orthopaedic specialists at the Hospital for Special Surgery,

New York.

GenotypingGenomic DNA was purified from the two parental couples and their

15 children after obtaining informed consent in accordance with

protocols approved by the Institutional Review Board of Weill

Cornell Medical College, and the Office of Human Research

Protection, Ministry of Health, Sultanate of Oman. DNA was

extracted from peripheral blood samples using commercial kits

(Qiagen, Volencia, CA). The Affymetrix 50K XbaI SNP Array was

used for genotyping as per the standard protocol described else-

where [Matsuzaki et al., 2004] by The University of Chicago

Functional Genomics Facility. Fine mapping was performed using

established microsatellite markers from the deCODE genetic map

identified through the NCBI Human Map Viewer (Build 35). PCR

reactions were performed in 10 ml volumes that contained 50 ng

genomic DNA; 0.25 U Platinum Taq DNA polymerase (Invitrogen

Carlsbad, CA); 1� PCR buffer, minus Mg; 1.5 mM MgCl2; 250 mM

primers; and 250 mM dNTPs. Amplification was performed using

the following PCR conditions (95�C� 75 sec, 10 cycles of

94�C� 45 sec, 55�C� 45 sec, 72�C� 75 sec, 30 cycles of

89�C� 45 sec, 55�C� 55 sec, 72�C� 45 sec followed by a final

extension of 72�C� 10 min). Amplified microsatellites were se-

quenced (ABI 3130 capillary DNA sequencer) and fragment analy-

sis was performed using the ABI GeneMapper v4.0 analysis

software.

Linkage AnalysisOf the 58,690 total SNPs, 2,186 were discarded from analysis

because they were not placed on the deCODE map or had a missing

rate >25%. Mendelian incompatibilities were identified using

PedCheck [O’Connell and Weeks, 1998]. Any markers that were

associated with Mendelian errors were dropped from subsequent

analysis. Linkage analysis was initially performed at 1 cM intervals

across each chromosome using Allegro 2.0 [Gudbjartsson et al.,

2005]. Parametric LOD scores assuming a fully penetrant autoso-

mal recessive mode of inheritance and a disease allele frequency

estimated at 1/10,000 were calculated.

RESULTS

The parents in each family were first cousins and both girls and boys

were affected, consistent with autosomal recessive inheritance

(Fig. 1A). The consanguinity, lack of cerebral calcifications in CT

scans of parents, and frequency of affected children supported a

fully penetrant autosomal recessive disorder, though an autosomal

dominant mutation with incomplete penetrance could not be

entirely ruled out.

History and Physical ExaminationSalient clinical features are summarized in Table I. Birth

parameters are noted below when available, but none of the affected

130 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

children were thought to be unusually small or underweight as

neonates.

Family 1Patient 1, the eldest affected, was a female with family history of

intracerebral calcifications in three other siblings. She was born at

full-term by normal vertex delivery with birth weight 2.8 kg (�1 SD

for length), length 50 cm, and occipito-frontal head circumference

(OFC) 30 cm (�2.8 SD), and had no perinatal problems. Her

language development was slow with a small vocabulary, and she

did not walk until 2 years, later than her older brothers. She was

noted to have short stature, thin build, and reduced vigour com-

pared to school mates, but had no major illnesses during childhood.

Her parents described school difficulties with poor academic

progress, but normal behavior. She complained of periodic head-

aches from age 15 years, and at about that age lost the ability to walk

fast or run. Brain calcifications were discovered at 17 years during

family screening for brain calcifications diagnosed in a younger

sister. She performed activities of daily living independently, but

her fine motor coordination was poor. When examined at 18 years,

her weight was 29 kg (�5 SD on CDC charts, estimated �3.5 SD in

the Omani population; hereafter�5/�3.5 SD), height 141 cm (�5/

�3.5 SD), and OFC 50 cm (�3 SD). She had no dysmorphic features

or skin changes, and her hair and nails were normal. Speech was

fluent with a normal cranial nerve exam. She walked without ataxia

or spasticity but was unable to run. Tendon reflexes were mildly

brisk.

Patient 2 was the product of a full-term normal delivery, birth

weight 2.9 kg (�1 SD for length), length 50 cm, and OFC 32 cm

(�2 SD). Like patient 1, her postnatal development was slow,

though her learning difficulties were less severe than her affected

FIG. 1. A: Pedigree of extended families 1 and 2. Brain CT images from Patient 1 (B) and Patient 4 (C) show extensive brain involvement with

calcifications of deep cerebellar nuclei, caudate, putamen, thalamus, and cerebral cortex. Cerebral cortical calcifications tend to occur at the

depths of sulci where U-fibers are located.

RAJAB ET AL. 131

sisters. At age 14 years her school evaluations indicated cognitive

skills commensurate with a 10-year-old. Examined at age 14 years,

she was not dysmorphic except that low set ears were noted. Skin,

hair, and nails were normal with no lesions. She weighed 17.3 kg

(�5/�3.5 SD), height measured 124 cm (�5/�3.5 SD), and OFC

47 cm (�3.3 SD).

Patient 3 was 11 years of age when examined. Slow growth and

developmental delay was noted from infancy. Her weight was

13.9 kg (�4.5/�3 SD), height 113 cm (�5/�3.5 SD), and OFC

47 cm (�3 SD). She was said to suffer severe learning difficulties,

and was unable to read; her speech was difficult to understand. She

was pleasant and cooperative with thin build and short stature.

There were no skin lesions or chilblains, but palmar erythema was

noted. Fundoscopic exam was normal with no retinal exudates and

normal retinal vessels. Cognitive difficulties were more pronounced

than her sisters and considerable dysarthria was apparent. She had

mild dyspraxia and her ability to imitate movements was limited.

Gait was slightly broad based with reduced arm swing. She was

unable to run or move fast. Deep tendon reflexes were brisk.

Patient 4 was seen at 7 years of age. She was the product of a full-

term normal delivery. Early infancy was uneventful, but an unusual

increase in head size was noted by age 3 months. A transcranial

ultrasound at age 6 months diagnosed right-sided hydrocephalus

and brain calcifications, and she appeared lethargic and was not

feeding well. Serological investigations for congenital infections

were negative. She had a ventriculo-peritoneal (VP) shunt inserted

at 8 months of age. Calcifications were evident on imaging at that

time. Her activity and growth improved post shunt. Seizures were

noted 1 year post VP shunt insertion and she was placed on

anticonvulsants. Like her sisters, she was slow to sit up, walk, and

talk. Mild to moderate learning difficulties were noted by her

parents. She assisted with dressing but was not independent. At

the age of 5 years she underwent an operative revision of the shunt

that was complicated by seizures. At age 6 years she

sustained a fracture of the left arm. At age 7 years her weight was

13.4 kg (�3.5/�2 SD), height 100 cm (�5/�3.5 SD), and OFC

47 cm (�2.3 SD). Cognitive development was delayed but social

interactions were normal. Speech was intelligible without dysar-

thria, and she was able to follow commands and imitate simple

motions. She had no gross motor abnormalities; tendon reflexes

were normal with flexor plantar responses.

Family 2Patient 5 was ascertained at age 28 years. He was unaware of brain

calcifications until the age of 28 when CT showed diffuse calcifi-

cations in bilateral frontal, temporal and parietal lobes, basal

ganglia, and cerebellum, but no hydrocephalus or brain atrophy.

He had no history of neonatal or perinatal complications, nor any

major illness or hospitalisations during childhood. Postnatal de-

velopment and growth were slower than his healthy siblings.

Learning difficulties were noted in school and he was not able to

complete intermediate school. He reported periodic headaches

occurring mostly in the mornings that responded only partially

to analgesics and were not associated with visual disturbance,

nausea, or vomiting. He had a history of hand tremors and

unprovoked sweating episodes. Several medical opinions were

sought for inability to gain weight despite heavy food intake but

no cause was found. He worked as a driver. When examined at

29 years, his weight was 40 kg (�3.5/�2 SD), height 153 cm (�3.5/

�2 SD), and OFC 53 cm (�2 SD), and he appeared emaciated with

poor muscle mass. His exam also showed no refractive errors,

normal vision, retina, and retinal vessels, normal muscle tone,

power, and tendon reflexes, and no cerebellar signs. Fine hand

tremors were noted at rest.

Patient 6 was the full-term product of a normal pregnancy with

birth weight 2.5 kg. At age 2 years she developed an acute febrile

illness. Lumbar puncture for assessment of possible meningitis was

complicated by seizure and decorticate rigidity, which may have

been due to downward herniation at the time of lumbar puncture.

TABLE I. Summary of Clinical Features of Affected Patients

Patient

1 2 3 4 5 6 7 8Widespread brain

calcificationsþ þ þ þ þ þ þ þ

Microcephaly(�2 to �3 SD)

þ þ þ þ þ þ þ þ

Postnatal growth delay þ þ þ þ þ þ þ þShort stature þ þ þ þ þ þ þ þThin build þ þ þ þ þ þ þ þPoor muscle mass þ þ þ þ þ þ þ þLearning difficulties þ þ þ þ þ NA þ �Reduced exercise

toleranceþ þ þ þ þ þ þ þ

Headaches þ þ þ þ þ NA þ �Bone fractures � � � þ � � � �Thin cortex bone þ þ þ þ þ NA þ þDelayed bone age þ þ þ þ þ NA þ þAnemia þ þ þ þ þ NA þ þ

132 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

Cerebrospinal fluid examination did not indicate meningitis. CT

scan showed widespread brain calcifications that were attributed to

congenital infection, though a TORCH screen at the age of 2 years

was negative. Spastic quadriplegia was noted on recovery. From that

time the patient suffered from severe spasticity and was bed-ridden

for life. She died at age 20 years due to pneumonia.

Patient 7 had a history of slow growth and developmental delay

with mild learning difficulties in school. He suffered headaches 2–3

times per week occurring at different times of the day and com-

plained of difficulty gaining weight despite adequate caloric intake.

Ascertained at the age of 18 years, he was a part time college student.

Examination showed a thin-built young man with poor muscle

mass, 38 kg (�4/�2.5 SD), height 155 cm (�3/�1.5 SD), and OFC

52 cm (�2 SD). Brain calcifications were present on his CT scan.

Patient 8 was born at full-term by normal vertex delivery with no

neonatal complications. She had no history of headaches, no visual

deficit, no history of fractures, and no anaemia. Examined at age 15

years, she was small in stature, of slight build, and poor muscle mass

with no skin lesions, normal hair, and nails. Her school perfor-

mance was said to be satisfactory. She weighed 30 kg (�4/�2.5 SD),

height measured 145 cm (�3/�1.5 SD), and OFC 51 cm (�2 SD),

and she had normal pubertal development. Neurological examina-

tion showed normal vision, retina, and retinal vessels. Like her other

affected siblings, she had reduced fine motor dexterity and slow

rapid alternating movements. A brain CT showed widespread

parenchymal calcifications and mild dilatation of lateral ventricles.

Laboratory InvestigationsBrain imaging of all eight children in families 1 and 2 were reviewed

and were remarkably similar. Brain CT scans obtained on the

parents in both families showed no calcifications. CT scans from

patients 1 and 4 are shown (Fig. 1). A brain CT of the eldest affected

daughter in Family 1 (patient 1, Fig. 1B) showed diffuse serpiginous

or globular calcifications involving frontal, temporal, parietal, and

occipital cortex, basal ganglia, thalamus, and deep cerebellar nuclei.

No significant brain atrophy was seen, and ventricular size appeared

normal. A brain CT obtained on her youngest sister (Patient 4) at

age 5 years (Fig. 1C) showed widespread curvilinear calcifications in

both cerebral hemispheres and small ventricles with shunt catheter

in place. Like her older siblings, the cortical calcifications occurred

predominantly at the base of sulci at the grey–white matter junc-

tion. An MRI obtained on Patient 1 (Fig. 2) showed signal voids

corresponding to the calcifications. The white matter appeared

intact with essentially normal signal intensity. Detailed retinal exam

in this girl showed no vasculopathy or angiomas (Fig. 2C).

The youngest girl in Family 1 (Patient 4), who received a VP

shunt, had CSF examined on three occasions and was found to have

a normal protein of 40 mg/dl and 2 cells per ml. In addition, Patient

6 (Family 2) had a lumbar puncture at age 2 during a febrile illness,

and the CSF protein and cell count were normal.

Skeletal X-rays on all four affected girls in Family 1 showed

intracranial calcification on skull films (Fig. 3). X-rays of hands and

long bones showed signs of thin cortical bone and osteopenia, with

the trabecular bone too readily visible (Fig. 3). Abdominal ultra-

sound showed normal liver, spleen, and kidneys, and no calcifica-

tions outside of brain were found in any of the affected individuals.

Additional normal laboratory values obtained on all affected

children included blood and platelet counts, arterial blood gases (on

Patients 1, 3, and 4), serum electrolytes, liver function tests (SGPT

and SGOT), alkaline phosphatase, calcium, parathormone, thyroid

function tests (TSH, T4), renal function tests (blood urea nitrogen,

creatinine), and glucose. In addition, HPLC studies excluded sickle

cell anemia and beta-thalassemia.

Recombination Frequency MappingAn affected only genome-wide linkage analysis was performed on

Family 1. The most significant scores were observed in two regions,

the first spanning 15 cM on chromosome 2 (214-229 cM) with a

LOD of 3.01 and the second spanning 11 cM on chromosome 12

(86-97 cM) with a LOD of 2.00. Fine-mapping using polymorphic

microsatellite markers between rs2373041 and rs10498202 on

2q35–q36.3 was performed in Families 1 and 2. The resulting peak

LOD score increased to 6.17 at D2S351 and D2S2390 on 2q36.2

(Fig. 4). Fine-mapping on 12q15-q21.31 excluded this locus (data

not shown).

DISCUSSION

All affected members of this extended, inbred family manifested a

syndrome of microcephaly, and cerebral calcifications associated

with developmental delay, small stature, and osteopenia. The

calcifications were striking, and involved the cerebral cortex, par-

ticularly the gray–white border in the depths of sulci where they

appeared to trace the U-fibers, as well as the basal ganglia and

cerebellum. While the ‘‘railroad track’’ appearance suggested cal-

cified vessels, no vascular abnormalities were seen on magnetic

resonance angiogram in the studies obtained on Patient 1. All

patients displayed poor weight gain from infancy and all were less

vigorous than their normal siblings. All affected individuals were

small with height and weight proportional to OFCs of�2 to�3 SD

below the mean. Although delayed, neurological milestones once

acquired were maintained and psychosocial development was

normal. School performance was suboptimal in all, but to variable

degrees, and fine dexterity was poor in most patients. One 18-year-

old displayed dyspraxia, brisk deep tendon reflexes, and lost her

ability to move fast and to run in her teens; another had fine hand

tremors at rest at the age of 28 years. Thin cortical bone with

prominent trabecular pattern and osteopenia on skeletal X-ray was

noted in all patients. However, only one child had a history of

traumatic fracture. Another child developed hydrocephalus, per-

haps due to the placement of calcifications, and seizures most likely

related to her VP shunt.

Among the inherited developmental disorders associated with

intracranial calcifications, perhaps four—AGS, CRMCC, Fried

syndrome, and CAII deficiency—most closely approximate the

pattern of calcifications seen here, though our patients’ syndrome

differs significantly from each of them. AGS is a severe autosomal

recessive encephalopathy characterized by stereotyped subacute

onset of irritability, inconsolable crying, loss of skills, and frequently

intermittent sterile fevers usually after the first few days but within

the first year of life, and subsequently severe mental retardation,

spasticity, and dystonia with no further progression, and recurrent

RAJAB ET AL. 133

chilblain lesions [Crow et al., 2006a,b; Rice et al., 2007]. A few AGS

patients are affected at birth with neonatal seizures, jitteriness, poor

feeding, thrombocytopenia, hepatosplenomegaly, and elevated

transaminases. Brain imaging shows progressive brain atrophy,

leukoencephalopathy, intracerebral calcifications that typically

spare the cortex (Table II). Chronic CSF lymphocytosis, increased

CSF alpha-interferon, and negative serologic investigations for

common prenatal infections are characteristic. The protean clinical

manifestations of AGS have led to several different names for this

syndrome, including familial infantile encephalopathy with intra-

cranial calcification and chronic cerebrospinal fluid lymphocytosis,

Cree encephalitis, and (incorrectly) pseudo-TORCH syndrome.

These are considered to be the same entity [Crow et al., 2003],

except that Briggs et al. [in press] have recently delineated a distinct

syndrome with polymicrogyria and intracerebral calcifications that

establish pseudo-TORCH as a separate entity.

At least four genes have been associated with AGS1–4, located on

chromosomes 3p21.3 [Crow et al., 2000, 2006a], 13q14 [Ali et al.,

2005], 11q13.2 [Crow et al., 2006b], and 19p13.13 [Crow et al.,

2006b], as summarized in [Rice et al., 2007]. The causative genes are

TREX1 (AGS1), encoding a 3’-5’ exonuclease, and RNASEH2A

(AGS2), RNASEH2B (AGS3), and RNASEH2C (AGS4), all of which

are involved in the degradation of DNA and RNA molecules [Crow

et al., 2006a,b]. It has been speculated that these genes are involved

in nucleic acid removal during apoptosis and that failure of this

process leads to activation of the innate immune system, accounting

for the features that resemble infection or autoimmune lupus-like

disease [Rice et al., 2007].

At least one AGS gene remains to be identified [Rice et al., 2007],

leaving open the possibility that the disorder reported here repre-

sents a new AGS locus on chromosome 2. However, the disorder

seen in this Omani family is distinct from any form of AGS. The

clinical course is significantly milder than those AGS families

described, although AGS2 patients have displayed slow progression

and milder symptoms [Crow et al., 2006b]. The pattern of calci-

fications differs (Table II) and no evidence of leukoencephalopathy

was present on MR imaging of Patient 1, which would have been

evident in AGS by that age. No hematological or biochemical

FIG. 2. Brain MRI and retinal photographs from patient 1. A,B: MR images show hypodense areas of calcification (e.g., arrows) that occur at the

base of a sulcus or in deep structures including caudate, putamen, thalamus, and dentate nuclei of the cerebellum. There is little or no white

matter dystrophic change. C: retinal photographs show a normal vascular pattern and absence of exudates.

134 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

abnormalities and no signs of organomegaly or cutaneous lesions

similar to chilblains were detected among affected individuals. The

critical interval identified by linkage analysis is large (15 cM), but

there are no known nucleotide exonucleases in the region. Thus, if

this extended family were affected by a disorder in the AGS

spectrum, the phenotype would expand the mild end of the

spectrum.

Fried syndrome is a rare X-linked syndrome characterized by

mental retardation, calcifications of the basal ganglia and deep

cerebellar nuclei, and facial dysmorphism in boys. Mutations of the

FIG. 3. Skeletal X-rays from Patient 1. A: skull film shows prominent intracranial calcifications (Arrows). B: Hand and (C) knee films show signs

of thinned cortical bone and prominent trabecular pattern consistent with osteopenia.

FIG. 4. Close-up of the chromosome 2 locus. Multipoint parametric LOD scores obtained from the fine-mapping (black line) analysis are shown.

Physical positions (UCSC Human Genome Browser May 2004), microsatellite markers and two genic regions harbouring 72 RefSeq genes

within the region of interest are depicted.

RAJAB ET AL. 135

adapter protein gene, AP1S2, on Xp22 were recently identified

[Saillour et al., 2007]. This locus was excluded in the family reported

here, and the phenotype differs due to the lack of growth retarda-

tion, microcephaly, cortical calcifications, and osteopenia. Another

syndrome with brain calcification is CAII deficiency, which is

characterized by osteopetrosis, renal tubular acidosis, intracranial

calcification, and mental retardation [Cumming and Ohlsson,

1985]. Although they share some features of brain calcification

(Table II), the CAII syndrome has increased bone density, and renal

acidosis, which are not present in patients of this extended Omani

family.

Perhaps a better fit for the Omani family is CRMCC or ‘‘Coats’

plus’’ syndrome [Crow et al., 2004]. This condition prominently

includes a retinal pathology known as Coats’ disease characterized

by the slow, insidious onset and progression of abnormal retinal

vascular permeability, and telangectasia [Tolmie et al., 1988]. It is

recognized as raised patches of yellow–white exudates beneath

normal vessels progressing to retinal detachment, cataract, glauco-

ma, phthisis bulbi, and blindness. Other features of CRMCC

include less severe neurological impairments than AGS, normal

life expectancy, and osteopenia with frequent fractures. Brain

imaging demonstrates a progressive leukoencephalopathy

and extensive calcifications (Table II), and a brain biopsy in one

patient showed local astrocytosis and calcification of small vessels

[Crow et al., 2004]. CRMCC was recently merged with

‘‘leukoencephalopathy with calcifications’’ or Labrune syndrome,

in which brain pathology demonstrates a small vessel microangi-

opathy, but in which retinal pathology may be lacking [Briggs et al.,

2008].

The phenotype in the Omani family reported here resembles

CRMCC in the mild to moderate cognitive impairment range, mild

motor impairment, growth deficiency, and osteopenia, and distri-

bution of intracerebral calcifications (Table II), but differs in many

regards. None had visual impairment and detailed retinal exam in

an 18-year-old affected girl and an affected man aged 28 years

showed no evidence of vasculopathy. Brain CT scans in eight

patients demonstrated no cerebral cysts, and the single brain MRI

showed no leukoencephalopathy or cerebral cysts. If the Omani

family disorder were in the CRMCC spectrum, the phenotype

would expand the mild end of the spectrum.

The critical interval encompassing the disease locus in this

extended family is large at 15 cM, due to consanguinity and the

relatively few informative markers available for this family. It is

difficult to speculate regarding candidate genes in this interval as

few genes have a demonstrated connection to intracerebral calcifi-

cation or obvious mechanistic explanation. There is no gene

in the region with known homology to RNAses or exonucleases,

the protein class associated with AGS, nor are there genes with

known roles in calcium homeostasis. Further refinement of the

locus will require identification of additional families with this

phenotype.

ACKNOWLEDGMENTS

Supported by NIH funded RO1 NS058721 to W.B.D. and P01

NS048120 to M.E.R.

REFERENCES

Ahn TG, Antonarakis SE, Kronenberg HM, Igarashi T, Levine MA. 1986.Familial isolated hypoparathyroidism: A molecular genetic analysis of 8families with 23 affected persons. Medicine (Baltimore) 65:73–81.

Aicardi J, Goutieres F. 1984. A progressive familial encephalopathy ininfancy with calcifications of the basal ganglia and chronic cerebrospinalfluid lymphocytosis. Ann Neurol 15:49–54.

Al Mane KA, Coates RK, McDonald P. 1996. Intracranial calcification inRaine syndrome. Pediatr Radiol 26:55–58.

Ali M, Highet LJ, Lacombe D, Goizet C, King MD, Tacke U, van der KnaapMS, Lagae L, Rittey C, Brunner HG, von Bokhoven H, Hamel B, Oade YA,Sanchis A, Desguerre I, Cau D, Mathieu N, Moutard ML, Lebon P, KumarD, Jackson AP, Crow YJ. 2005. A second locus for Aicardi-Goutieressyndrome at chromosome 13q 14–21. J Med Genet 43:440–450.

Baba Y, Broderick DF, Uitti RJ, Hutton ML, Wszolek ZK. 2005. Here-dofamilial brain calcinosis syndrome. Mayo Clin Proc 80:641–651.

Baumert T, Kleber G, Schwarz J, Stabler A, Lamerz R, Mann K. 1993.Reversible hyperkinesia in a patient with autoimmune polyglandularsyndrome type I. Clin Investig 71:924–927.

Briggs TA, Abdel-Salam GM, Balicki M, Baxter P, Bertini E, Bishop N,Browne BH, Chitayat D, Chong WK, Eid MM, Halliday W, Hughes I,Klusmann-Koy A, Kurian M, Nischal KK, Rice GI, Stephenson JB, SurteesR, Talbot JF, Tehrani NN, Tolmie JL, Toomes C, van der Knaap MS, CrowYJ. 2008. Cerebroretinal microangiopathy with calcifications and cysts(CRMCC). Am J Med Genet Part A 146A:182–190.

Briggs TA, Wolf NI, D’arrigo S, Ebinger F, Harting I, Dobyns WB,Livingston JH, Rice GI, Crooks D, Rowland-Hill CA, Squier W, StoodleyN, Pilz DT, Crow YJ. In press. Band-like intracranial calcification withsimplified gyration and polymicrogyria; another recognizable ‘pseudo-torch’ phenotype. Am J Med Genet Part A (in press).

TABLE II. Location of Intracerebral Calcifications in Five Syndromes

Calcification

Syndrome

Fried AGS CRMCC CAII This reportCortex-superficial WM (U-fibers) þ þ þDeep and periventricular WM þ þBasal ganglia and thalamus þ þ þ þ þCerebellar dentate nuclei þ þ þ (þ) þþ, typical location; (þ), occasional location; CAII, carbonic anhydrase II deficiency; Fried, Fried syndrome; WM, white matter changes.

136 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

Burn J, Wickramasinghe HT, Harding B, Baraitser M. 1986. A syndromewith intracranial calcification and microcephaly in two sibs, resemblingintrauterine infection. Clin Genet 30:112–116.

Crow YJ, Jackson AP, Roberts E, van Beusekom E, Barth P, Corry P, FerrieCD, Hamel BC, Jayatunga R, Karbani G, K�alm�anchey R, Kelemen A, KingM, Kumar R, Livingstone J, Massey R, McWilliam R, Meager A, Rittey C,Stephenson JB, Tolmie JL, Verrips A, Voit T, van Bokhoven H, BrunnerHG, Woods CG. 2000. Aicardi-Goutieres syndrome displays geneticheterogeneity with one locus (AGS1) on chromosome 3p21. Am J HumGenet 67:213–221.

Crow YJ, Black DN, Ali M, Bond J, Jackson AP, Lefson M, Michaud J,Roberts E, Stephenson JB, Woods CG, Lebon P. 2003. Cree encephalitis isallelic with Aicardi-Goutieres syndrome: Implications for the pathogen-esis of disorders of interferon alpha metabolism. J Med Genet40:183–187.

Crow YJ, McMenamin J, Haenggeli CA, Hadley DM, Tirupathi S, TreacyEP, Zuberi SM, Browne BH, Tolmie JL, Stephenson JB. 2004. Coats’ plus:A progressive familial syndrome of bilateral Coats’ disease, characteristiccerebral calcification, leukoencephalopathy, slow pre- and post-natallinear growth and defects of bone marrow and integument. Neuro-pediatrics 35:10–19.

Crow YJ, Hayward BE, Parmar R, Robins P, Leitch A, Ali M, Black DN, vanBokhoven H, Brunner HG, Hamel BC, Corry PC, Cowan FM, Frints SG,Klepper J, Livingston JH, Lynch SA, Massey RF, Meritet JF, Michaud JL,Ponsot G, Voit T, Lebon P, Bonthron DT, Jackson AP, Barnes DE,Lindahl T. 2006a. Mutations in the gene encoding the 3’-5’ DNAexonuclease TREX1 cause Aicardi-Goutieres syndrome at the AGS1locus. Nat Genet 38:917–920.

Crow YJ, Leitch A, Hayward BE, Garner A, Parmar R, Griffith E, Ali M,Semple C, Aicardi J, Babul-Hirji R, Baumann C, Baxter P, Bertini E,Chandler KE, Chitayat D, Cau D, D�ery C, Fazzi E, Goizet C, King MD,Klepper J, Lacombe D, Lanzi G, Lyall H, Mart�ınez-Fr�ıas ML, Mathieu M,McKeown C, Monier A, Oade Y, Quarrell OW, Rittey CD, Rogers RC,Sanchis A, Stephenson JB, Tacke U, Till M, Tolmie JL, Tomlin P, Voit T,Weschke B, Woods CG, Lebon P, Bonthron DT, Ponting CP, Jackson AP.2006b. Mutations in genes encoding ribonuclease H2 subunits causeAicardi-Goutieres syndrome and mimic congenital viral brain infection.Nat Genet 38:910–916.

Cumming WA, Ohlsson A. 1985. Intracranial calcification in children withosteopetrosis caused by carbonic anhydrase II deficiency. Radiology157:325–327.

Farley TJ, Ketonen LM, Bodensteiner JB, Wang DD. 1992. Serial MRIand CT findings in infantile Krabbe disease. Pediatr Neurol 8:455–458.

Geschwind DH, Loginov M, Stern JM. 1999. Identification of a locus onchromosome 14q for idiopathic basal ganglia calcification (Fahr disease).Am J Hum Genet 65:764–772.

Gudbjartsson DF, Thorvaldsson T, Kong A, Gunnarsson G, Ingolfsdottir A.2005. Allegro version 2. Nat Genet 37:1015–1016.

Hirano M, Pavlakis SG. 1994. Mitochondrial myopathy, encephalopathy,lactic acidosis, and strokelike episodes (MELAS): Current concepts. JChild Neurol 9:4–13.

Juppner H, Schipani E, Bastepe M, Cole DE, Lawson ML, Mannstadt M,Hendy GN, Plotkin H, Koshiyama H, Koh T, Crawford JD, Olsen BR,Vikkula M. 1998. The gene responsible for pseudohypoparathyroidismtype Ib is paternally imprinted and maps in four unrelated kindreds tochromosome 20q13.3. Proc Natl Acad Sci USA 95:11798–11803.

Kan AE, Kozlowski K. 1992. New distinct lethal osteosclerotic bonedysplasia (Raine syndrome). Am J Med Genet 43:860–864.

Lebon P, Badoual J, Ponsot G, Goutieres F, Hemeury-Cukier F, Aicardi J.1988. Intrathecal synthesis of interferon-alpha in infants with progressivefamilial encephalopathy. J Neurol Sci 84:201–208.

Matsuzaki H, Dong S, Loi H, Di X, Liu G, Hubbell E, Law J, Berntsen T,Chadha M, Hui H, Yang G, Kennedy GC, Webster TA, Cawley S, WalshPS, Jones KW, Fodor SP, Mei R. 2004. Genotyping over 100,000 SNPs ona pair of oligonucleotide arrays. Nat Methods 1:109–111.

O’Connell JR, Weeks DE. 1998. PedCheck: A program for identification ofgenotype incompatibilities in linkage analysis. Am J Hum Genet63:259–266.

Patten JL, Johns DR, Valle D, Eil C, Gruppuso PA, Steele G, Smallwood PM,Levine MA. 1990. Mutation in the gene encoding the stimulatory Gprotein of adenylate cyclase in Albright’s hereditary osteodystrophy. NEngl J Med 322:1412–1419.

Reardon W, Hockey A, Silberstein P, Kendall B, Farag TI, Swash M,Stevenson R, Baraitser M. 1994. Autosomal recessive congenital intra-uterine infection-like syndrome of microcephaly, intracranial calcifica-tion, and CNS disease. Am J Med Genet 52:58–65.

Rice G, Patrick T, Parmar R, Taylor CF, Aeby A, Aicardi J, Artuch R,Montalto SA, Bacino CA, Barroso B, Baxter P, Benko WS, Bergmann C,Bertini E, Biancheri R, Blair EM, Blau N, Bonthron DT, Briggs T, BruetonLA, Brunner HG, Burke CJ, Carr IM, Carvalho DR, Chandler KE,Christen HJ, Corry PC, Cowan FM, Cox H, D’Arrigo S, Dean J, De LaetC, De Praeter C, Dery C, Ferrie CD, Flintoff K, Frints SG, Garcia-CazorlaA, Gener B, Goizet C, Goutieres F, Green AJ, Guet A, Hamel BC, HaywardBE, Heiberg A, Hennekam RC, Husson M, Jackson AP, Jayatunga R, JiangYH, Kant SG, Kao A, King MD, Kingston HM, Klepper J, van der KnaapMS, Kornberg AJ, Kotzot D, Kratzer W, Lacombe D, Lagae L, LandrieuPG, Lanzi G, Leitch A, Lim MJ, Livingston JH, Lourenco CM, Lyall EG,Lynch SA, Lyons MJ, Marom D, McClure JP, McWilliam R, MelanconSB, Mewasingh LD, Moutard ML, Nischal KK, Ostergaard JR, PrendivilleJ, Rasmussen M, Rogers RC, Roland D, Rosser EM, Rostasy K, RoubertieA, Sanchis A, Schiffmann R, Scholl-Burgi S, Seal S, Shalev SA, CorcolesCS, Sinha GP, Soler D, Spiegel R, Stephenson JB, Tacke U, Tan TY, Till M,Tolmie JL, Tomlin P, Vagnarelli F, Valente EM, Van Coster RN, Van derAa N, Vanderver A, Vles JS, Voit T, Wassmer E, Weschke B, WhitefordML, Willemsen MA, Zankl A, Zuberi SM, Orcesi S, Fazzi E, Lebon P,Crow YJ. 2007. Clinical and molecular phenotype of Aicardi-Goutieressyndrome. Am J Hum Genet 81:713–725.

Saillour Y, Zanni G, Des Portes V, Heron D, Guibaud L, Iba-Zizen MT,Pedespan JL, Poirier K, Castelnau L, Julien C, Franconnet C, Bonthron D,Porteous ME, Chelly J, Bienvenu T. 2007. Mutations in the AP1S2 geneencoding the sigma 2 subunit of the adaptor protein 1 complex areassociated with syndromic X-linked mental retardation with hydroceph-alus and calcifications in basal ganglia. J Med Genet 44:739–744.

Sly WS, Whyte MP, Sundaram V, Tashian RE, Hewett-Emmett D, GuibaudP, Vainsel M, Baluarte HJ, Gruskin A, Al-Mosawi M, Sakati N, Ohlsson A.1985. Carbonic anhydrase II deficiency in 12 families with the autosomalrecessive syndrome of osteopetrosis with renal tubular acidosis andcerebral calcification. N Engl J Med 313:139–145.

Tolmie JL, Browne BH, McGettrick PM, Stephenson JB. 1988. A familialsyndrome with coats’ reaction retinal angiomas, hair and nail defects andintracranial calcification. Eye 2:297–303.

Tolmie JL, Shillito P, Hughes-Benzie R, Stephenson JB. 1995. The Aicardi-Goutieres syndrome (familial, early onset encephalopathy with calcifi-cations of the basal ganglia and chronic cerebrospinal fluidlymphocytosis). J Med Genet 32:881–884.

Woody RC, Brewster MA, Glasier C. 1989. Progressive intracranial calcifi-cation in dihydropteridine reductase deficiency prior to folinic acidtherapy. Neurology 39:673–675.

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